Some thiazole derivatives bearing dithiocarbamic acid esters were synthesized in order to investigate their anticandidal activity and cytotoxicity. The structures of the obtained final compounds (6a–j) were confirmed by spectral data (IR, 1H NMR, 13C NMR, and MS) and elemental analysis. The anticandidal activity of the compounds was determined (6a–j) using the microbroth dilution method and their cytotoxicity was evaluated according to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay against normal cells. Contrary to expectations, weak antifungal activity was observed with IC50 values ranging between 30 and 403 µg/mL.
Trang 1⃝ T¨UB˙ITAK
doi:10.3906/kim-1312-62
h t t p : / / j o u r n a l s t u b i t a k g o v t r / c h e m /
Research Article
Synthesis, anticandidal activity, and cytotoxicity of some thiazole derivatives with
dithiocarbamate side chains
Leyla YURTTAS ¸1, ∗, Yusuf ¨ OZKAY1, Fatih DEM˙IRC˙I2, Gamze G ¨ OGER2,
S ¸afak ULUSOYLAR YILDIRIM3, Usama ABU MOHSEN4, ¨ Omer ¨ OZT ¨ URK1,
Zafer Asım KAPLANCIKLI1,5
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eski¸sehir, Turkey
2
Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, Eski¸sehir, Turkey
3
Department of Pharmacology, Faculty of Pharmacy, Anadolu University, Eski¸sehir, Turkey
4
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Gaza, Palestine
5Department of Pharmaceutical Chemistry, Graduate School of Health Sciences, Anadolu University,
Eski¸sehir, Turkey
Received: 26.12.2013 • Accepted: 26.03.2014 • Published Online: 15.08.2014 • Printed: 12.09.2014
Abstract: Some thiazole derivatives bearing dithiocarbamic acid esters were synthesized in order to investigate their anticandidal activity and cytotoxicity The structures of the obtained final compounds (6a–j) were confirmed by spectral
data (IR, 1H NMR, 13C NMR, and MS) and elemental analysis The anticandidal activity of the compounds was
determined (6a–j) using the microbroth dilution method and their cytotoxicity was evaluated according to the MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay against normal cells Contrary to expectations, weak antifungal activity was observed with IC50 values ranging between 30 and 403 µ g/mL.
Key words: Thiazole, dithiocarbamate, anticandidal activity, cytotoxicity
1 Introduction
Candidiasis encompasses infections that range from superficial, such as oral thrush and vaginitis, to systemic and potentially life-threatening diseases particularly in patients undergoing anticancer chemotherapy, organ transplants, or long treatment with antimicrobial agents and in patients with AIDS because of immune system suppression Such a broad range of infections and development of resistance to currently available antifungal
which are an important class of sulfur-containing compounds, were also described as herbicides and fungicides,
last decade, dithiocarbamate moiety combined with different heterocyclic ring systems was studied widely,
compounds are known to act as inhibitors of enzymes and have a profound effect on biological systems, because
of their strong metal-binding capacity The well-known thiocarbamate class of antifungal drug tolciclate (I) and the fungicidal active plant defense agent brassinin (II) are famous sulfurated compounds Additionally,
∗Correspondence: lyurttas@anadolu.edu.tr
Trang 2rhodanine (III) and its derivatives are the other dithiocarbamate-including molecules known for their ability to
inhibit fungal protein mannosyl transferase 1 (PMT1), which plays a key role in the biosynthesis of the fungal
Figure The chemical structures of the tolciclate (I), brassinin (II), and rhodanine (III).
Thiazoles have a prominent position among heterocycles and they can be obtained from microbial and
(anti-neoplastic agents), ritonavir (anti-HIV drug), fanetizole and meloxicam (anti-inflammatory agents), nizatidine
peni-cillin (antibiotic) Besides these bioactive compounds, there are a lot of studies about thiazole derivatives with
According to the foregoing literature survey, we now report the synthesis of the dithiocarbamic acid
derivatives of N -[4-(2-methyl-4-thiazolyl)phenyl]acetamide structure with potential anticandidal activity and
cytotoxicity in this study
2 Results and discussion
The present study was undertaken to synthesize some thiazole derivatives bearing dithiocarbamic acid ester and
to investigate their anticandidal activity and cytotoxicity The target compounds were obtained in multistep organic synthesis as shown in the Scheme The initial compound 4-aminoacetophenone in a TEA/THF mixture
was acetylated with chloroacetyl chloride to obtain 4-(acetylamino)acetophenone (1); then compound 1 in AcOH
was brominated to obtain N -[4-(2-bromoacetyl)phenyl]acetamide (2) The obtained amide compound (2) was reacted with thioacetamide to give N -[4-(2-methyl-4-thiazolyl)phenyl]acetamide (3) After hydrolysis of the
acetyl group on amino moiety, compound 4-(2-methyl-4-thiazolyl)aniline (4) was synthesized, which was then
acetylated with chloroacetyl chloride to obtain 2-chloro- N -[4-(2-methyl-4-thiazolyl)phenyl]acetamide (5) In
the final step, compound 5 was reacted with appropriate dithiocarbamate salts to give the final compounds (6a–6j) Compound 6c (2-[[4-(2-methyl-4-thiazolyl)phenyl]amino]-2-oxoethyl morpholine-4-carbodithioate) was
synthesized and registered with the chemical abstract service before, but there are no scientific data about the molecule and so we included this compound in our research
The structures of the synthesized compounds were elucidated by spectral data and elemental analysis, and significant stretching bands in the IR spectra were observed in the expected regions Stretching bands
NMR spectra of the compounds, methyl protons at the second position of the thiazole ring and N–H protons
Trang 3Scheme The synthesis of the compounds (6a–j) Reagents: (i) acetyl chloride, TEA, THF, 0–5 ◦C; (ii) Br2, AcOH; (iii) thioacetamide, EtOH, r.t (iv) 10% HCl, EtOH, reflux; (v) chloroacetyl chloride, TEA, THF, r.t.; (vi) appropriate
sodium salts of N, N -disubstituted dithiocarbamic acids, K2CO3, acetone, reflux
the thiazole ring was observed at about 7.23–7.27 ppm and as 2 singlets that are thought to be due to magnetic
ppm as singlets and protons of the cyclic structures were seen at 1.73 ppm and 4.65 ppm as broad singlets,
Trang 4commonly The other peaks belonging to aromatic and aliphatic protons were observed in the estimated areas.
peaks at 191–197 ppm C=N carbons of the thiazole ring were recorded at 156–157 ppm The mass spectra (EI-MS) of the compounds showed (M+1) peaks in agreement with their molecular weight Elemental analysis results for C, H, and N elements were satisfactory within calculated values of the compounds
The target compounds 6a–j were screened for their in vitro anticandidal activity against 7 candida
species, including standard strains and clinical isolates MIC is defined as the concentration of the compound required to give complete inhibition of bacterial growth and MICs of the synthesized compounds along with the reference drug ketoconazole are given The results provided in Table 1 indicate that most of the prepared
compounds displayed broad antifungal spectra with MIC values ranging from 62.5 to 125 µ g/mL against all the
tested strains Among all evaluated strains, the compounds 6a, 6b, 6c, 6f, and 6g had anticandidal activity
lower than that of the standard drug Compound 6a inhibited all species at a concentration of 62.5 µ g/mL except against C tropicalis Both of the compounds 6b and 6c displayed anticandidal activity against C.
albicans (ATCC 90028) and C glabrata (isolate 1) at the same concentrations (62.5 µ g/mL) Compounds were
against NIH/3T3 cells are shown in Table 2 The biological study indicated that compound 6f possessed the
highest cytotoxicity, with a value of about 30 µ g/mL, whereas compound 6i exhibited the lowest cytotoxicity,
with a value of about 330 µ g/mL, against NIH/3T3 cells.
Table 1 Anticandidal activity of the compounds (MIC in µ g/mL).
6a 62.5 62.5 62.5 125 62.5 62.5 62.5
6b 125 62.5 62.5 125 125 62.5 125
6c 62.5 62.5 125 125 125 62.5 125
6f 125 125 125 62.5 62.5 125 62.5
6g 62.5 125 125 125 125 62.5 62.5
Ref.: Ketoconazole, ND: Not defined
A: C albicans (isolate, obtained from Department of Microbiology, Faculty of Medicine, Osmangazi University, Eski¸sehir, Turkey), B: C glabrata (isolate 1, obtained from Department of Microbiology, Faculty of Medicine, Osmangazi University, Eski¸sehir, Turkey), C: C utilis (NRRLY-900), D: C tropicalis (NRRLY-12968), E: C krusei (NRRLY-7179), F: C albicans (ATCC 90028), G: C glabrata (isolate 2, obtained from Department of Microbiology, Faculty of Medicine,
Osmangazi University, Eski¸sehir, Turkey)
3 Conclusion
In this study, we report the synthesis, spectral studies, and biological evaluation of some thiazole derivatives
bearing dithiocarbamic acid ester (6a–j) The structures proposed for the synthesized compounds (6a–j) are
Trang 5well supported by spectroscopic data and elemental analysis Some of the final compounds (6a, 6b, 6c, 6f, and 6g) were evaluated for their anticandidal activity and they exhibited weak activity against all tested strains The cytotoxicity of the compounds was also studied and compounds 6c, 6d, 6h, and 6i displayed the lowest
cytotoxicity against NIH/3T3 cells
Table 2 In vitro cytotoxicity of the compounds.
aCytotoxicity of the compounds to mouse fibroblast (NIH/3T3) cell line Incubation for 24 h IC50 is the drug concentration required to inhibit 50% of the cell growth The values represent mean ± standard deviation of triplicate
determinations
4 Experimental
All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) All melting points (mps) were deter-mined by Electrothermal 9100 digital melting point apparatus (Electrothermal, Essex, UK) and are uncorrected All the reactions were monitored by thin-layer chromatography (TLC) using Silica Gel 60 F254 TLC plates (Merck KGaA, Darmstadt, Germany) Spectroscopic data were recorded with the following instruments: IR, Shimadzu 8400S spectrophotometer (Shimadzu, Tokyo, Japan); NMR, VARIAN Mercury 400 FT spectrometer
AB Sciex-3200 Q-TRAP LC/MS/MS system (Applied Biosystems Co., MA, USA)
4.1 4’-Acetaminoacetophenone (1)
4’-Aminoacetophenone (0.05 mol, 6.75 g) and triethylamine (0.06 mol, 8.34 mL) were dissolved in THF (100
solution The reaction mixture was stirred for 1 h at room temperature After evaporation of solvent, the
4.2 4-(2-Bromoacetyl)acetanilide (2)
Compound 1 (0.04 mol, 7.08 g) and HBr (0.5 mL) were dissolved in acetic acid (30 mL) and bromine (0.044
mol, 2.27 mL) was added dropwise at room temperature After completion of the addition of bromine, the reaction mixture was stirred for 1 h and then poured into ice-water (100 mL) The precipitated product was
Trang 6185–187 ◦C).22 IR: (KBr) ν max (cm−1) : 3363 (amide N–H), 3062 (aromatic C–H), 1698 (ketone C=O), 1665
(amide C=O), 1378–1196 (C–N), and 848 (1,4-disubstituted benzene)
4.3 4-(2-Methyl-4-thiazolyl)acetanilide (3)
Compound 2 (0.03 mol, 7.68 g) and thioacetamide (0.03 mol, 2.25 g) in ethanol (100 mL) were stirred at room
temperature for 48 h The precipitated product was filtered, dried, and recrystallized from ethanol Yield: 78%;
(amide C=O), 1367–1211 (C–N), and 843 (1,4-disubstituted benzene)
4.4 4-(2-Methyl-4-thiazolyl)aniline (4)
Compound 3 (0.025 mol, 5.8 g) was refluxed in 10% HCl (100 mL) for 1 h The mixture was cooled down,
(1,4-disubstituted benzene)
4.5 2-Chloro-N -[4-(2-methyl-4-thiazolyl)phenyl]acetamide (5)
Chloroacetyl chloride (0.02 mol, 1.6 mL) was added dropwise over 15 min to a magnetically stirred solution of
compound 4 (0.02 mol, 3.8 g) and triethylamine (0.02 mol, 2.8 mL) in dry THF (15 mL) After completion
of the reaction, the solvent was evaporated under reduced pressure Water was added to wash the resulting
solid and the mixture was filtered, dried, and recrystallized from ethanol to give compound 5 Yield: 83%; mp
(d, 2H, J = 8.1 Hz, Ar–H), and 10.50 (s, 1H, N–H).
4.6 General methods for synthesis of compounds 6a–j
Compound 5 (0.001 mol) was stirred with appropriate sodium salts of dithiocarbamic acids (0.0011 mol) in
acetone for 3 h The precipitated product was filtered and washed with water
4.6.1 2-[[4-(2-Methyl-4-thiazolyl)phenyl]amino]-2-oxoethyl diethylcarbamodithiodate (6a)
167.45, 194.90 MS (ES+): m/z 380
Trang 74.6.2 2-[[4-(2-Methyl-4-thiazolyl)phenyl]amino]-2-oxoethyl pyrrolidine-1-carbodithiodate (6b)
154.92, 166.00, 167.39, 191.87 MS (ES+): m/z 378
4.6.3 2-[[4-(2-Methyl-4-thiazolyl)phenyl]amino]-2-oxoethyl morpholine-4-carbodithioate (6c)
166.62, 196.471 MS (ES+): m/z 394
4.6.4 2-[[4-(2-Methyl-4-thiazolyl)phenyl]amino]-2-oxoethylthiomorpholine-4-carbodithioate (6d)
40.73, 54.01, 56.03, 111.85, 120.08, 127.10, 130.94, 137.88, 154.83, 166.05, 166.83, 196.02 MS (ES+): m/z 410
4.6.5 2-[[4-(2-Methyl-4-thiazolyl)phenyl]amino]-2-oxoethyl piperidine-1-carbodithioate (6e)
167.16, 194.41 MS (ES+): m/z 390
4.6.6 2-[[4-(2-Methyl-4-thiazolyl)phenyl]amino]-2-oxoethyl 4-methylpiperazine-1-carbodithioate (6f )
154.85, 166.07, 167.11, 196.02 MS (ES+): m/z 407
Trang 84.6.7 2-[[4-(2-Methyl-4-thiazolyl)phenyl]amino]-2-oxoethyl 4-ethyllpiperazine-1-carbodithioate (6g)
111.84, 120.11, 127.08, 130.84, 137.98, 154.86, 166.09, 167.15, 195.79 MS (ES+): m/z 421
4.6.8 2-[[4-(2-Methyl-4-thiazolyl)phenyl]amino]-2-oxoethyl 4-benzylpiperazine-1-carbodithioate (6h)
129.33, 130.86, 137.17, 137.77, 154.67, 165.79, 166.89, 195.61 MS (ES+): m/z 483
4.6.9 2-[[4-(2-Methyl-4-thiazolyl)phenyl]amino]-2-oxoethyl 4-(pyrimidin-2-yl)piperazine-1-carbo-dithioate (6i)
J = 4.8 Hz, 2H, Ar–H), 9.13 (s, 1H, N–H). 13C NMR (100 MHz, CDCl3) δ 19.32, 40.34, 42.94, 50.10, 52.16,
110.91, 111.58, 119.86, 126.86, 130.66, 137.70, 154.62, 157.84, 161.16, 165.81, 166.71, 196.27 MS (ES+): m/z 471
4.6.10 Bis{2-[[4-(2-methyl-4-thiazolyl)phenyl]amino]-2-oxoethyl}
piperazine-1,4-bis(carbodithio-ate) (6j)
139.32, 154.24, 165.89, 166.05, 195.60 MS (ES+): m/z 699
4.7 Anticandidal activity assay
Anticandidal activity of the final compounds was evaluated by the broth microdilution method according to the
Trang 9were as follows: C albicans (isolate, obtained from Department of Microbiology, Faculty of Medicine, Osmangazi University, Eski¸sehir, Turkey), C glabrata (isolate 1, obtained from Department of Microbiology, Faculty of Medicine, Osmangazi University, Eski¸sehir, Turkey), C utilis (NRRLY-900), C tropicalis (NRRLY-12968), C.
krusei (NRRLY-7179), C albicans (ATCC 90028), and C glabrata (isolate 2, obtained from Department of
Microbiology, Faculty of Medicine, Osmangazi University, Eski¸sehir, Turkey) Ketoconazole was used as positive control and the results (MIC values) are shown in Table 1
4.8 Cytotoxicity assay
Cytotoxic properties of the compounds were determined by the method mentioned in the literature using mouse
2 The procedure was realized using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
per well) All the compounds were dissolved in DMSO individually and added to culture wells at varying
concentrations (0.5–500 µ g/mL); the highest final DMSO concentration was under 0.1% After 24 h of drug
a 3-h incubation period was maintained in the same conditions Purple formazan occurred at the end of the process, which is the reduction product of MTT agent by the mitochondrial dehydrogenase enzyme of intact cells Formazan crystals were dissolved in 100 mL of DMSO and the absorbance was read by ELISA reader (OD 570 nm) The percentage of viable cells was calculated based on the medium control
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